Methods for enhancing the potency of the immune checkpoint inhibitors

ABSTRACT

The present invention relates to methods for enhancing the potency of the immune checkpoint inhibitors. In particular, the present invention relates to a method for enhancing the potency of an immune checkpoint inhibitor administered to a subject as part of a treatment regimen, the method comprising administering a pharmaceutically effective amount of a SK1 inhibitor to a subject in combination with the immune checkpoint inhibitor.

FIELD OF THE INVENTION

The present invention relates to methods for enhancing the potency ofthe immune checkpoint inhibitors.

BACKGROUND OF THE INVENTION

The ability of the immune system to detect and eliminate cancer wasfirst proposed over 100 years ago. Since then, T cells reactive againsttumor-associated antigens have been detected in the blood of patientswith many different types of cancers, suggesting a role for the immunesystem in fighting cancer. However, tumors can escape host immunity bymanipulating the tumor microenvironment and driving immunosuppression,meaning that patients cannot mount a potent enough immune response tofully eliminate cancer cells. The goal of immunotherapy is to restore oraugment antitumor immune responses. An increased understanding of tumorimmunology has led to the identification of novel targets for newimmune-based approaches, including a group of cell-surface moleculesknown as immune checkpoint proteins. In particular, monoclonalantibodies inhibiting CTLA-4 (ipilimumab) or PD-1 (nivolumab,pembrolizumab) have demonstrated significant efficacy in the treatmentof metastatic melanoma, promoting high response rate and long-lastingtumor control. Despite promising results, about 40% of patients do notdisplay therapeutic response and a significant proportion of respondersexperience tumor relapse in the 2 years following treatment induction.Moreover, recent clinical trials combining BRAF and checkpointinhibitors have shown high liver toxicity for patients with BRAF-mutatedmelanoma. Accordingly, development of novel therapeutic strategies isthus urgently needed in order to enhance the potency of the immunecheckpoint inhibitor.

Sphingolipid metabolites, including ceramide, ceramide 1-phosphate,sphingosine, and sphingosine 1-phosphate (S 1P), have emerged asbioactive signalling molecules that regulate cell motility,differentiation, proliferation and survival as well as angiogenesis,inflammation and immunity. It was recently demonstrated an increasedproduction of S1P in melanoma cells (2, 3). This bioactive sphingolipidmetabolite is produced mainly by sphingosine kinase 1 (SK1), which isoverexpressed in human melanoma tumors compared to nevi (2). In manytumors, S 1P conveys oncogenic signals as an intracellular secondmessenger and/or through the stimulation of a family of G-proteincoupled receptors (S1PR1-5) expressed both on cancer cells and theirsurrounding microenvironment (4, 5). In melanoma tumors, dysregulationof S1P production in cancer cells elicits a fibrotic response in thetumor microenvironment, which in turn stimulates melanoma cell migration(2). Additionally, treatment of mice with the S1P receptor modulatorFTY720, which renders cells unresponsive to S1P activation bysequestering S1PR1 internally, reduced melanoma progression byinhibiting tumor vascularization (6). These findings illustrate theparacrine action of melanoma cell-exported S1P through S1PRs ontumor-stroma interactions. However, recent studies demonstrate that theSK1/S1P/S1PR axis plays an essential role in inflammation-associatedcancer development (7). Indeed, shRNA-based downregulation of SK1 orS1PR1 has been shown to block the persistent activation of thetranscription factor STAT3 and the level of proinflammatory cytokinesand reduce cancer progression in mouse models of inflammation (8, 9). Inaddition, S1P contributes to trafficking and effector functions oflymphocytes and other hematopoietic cells (10). However, the prior artdoes not suggest SK1 inhibition could enhance the potency of the immunecheckpoint inhibitors.

SUMMARY OF THE INVENTION

The present invention relates to methods for enhancing the potency ofthe immune checkpoint inhibitors. In particular, the present inventionis defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

The inventors demonstrated that interfering with sphingolipid metabolismefficiently impairs tumor progression in pre-clinical melanoma model andenhances anti-tumor immune response obtained with the immune checkpointinhibitor. In particular, the inventors observed that SK1 downregulationenhances proliferation and activation of CD8+ T cells within the tumors.Of great interest is the finding that SK1 knockdown in melanoma enhancesthe expression of CTLA-4 and PD-1 on CD8+ TIL, which are bothup-regulated upon T cell activation and exert potent negative feedbackloop on T cell activation. The latter observation highlights for thefirst time that melanoma SK1 impairs CD8+ T cell-dependent immuneresponse. However, the upregulation of both PD-1 and CTLA-4 on CD8+ Tcells is likely involved in the melanoma immune escape following SK1knockdown observed at latter time points. Thus, targeting melanoma SK1is unlikely sufficient to trigger total tumor regression. Collectively,the data prompted the inventors to investigate the combination of SK1inhibition with the inhibition of immune checkpoints and demonstratethat said combination provides synergistic anti-cancer immune responses.

Accordingly the first object of the present invention relates to amethod of enhancing the proliferation and activation of tumorinfiltrating CD8+ T cells in a patient suffering from cancer comprisingadministering to the patient a therapeutically effective amount of a SK1inhibitor.

As used herein, the term “CD8+ T cell” has its general meaning in theart and refers to a subset of T cells which express CD8 on theirsurface. They are MHC class I-restricted, and function as cytotoxic Tcells. “CD8+ T cells” are also called cytotoxic T lymphocytes (CTL),T-killer cells, cytolytic T cells, or killer T cells. CD8 antigens aremembers of the immunoglobulin supergene family and are associativerecognition elements in major histocompatibility complex classI-restricted interactions. As used herein, the term “tumor infiltratingCD8+ T cell” refers to the pool of CD8+ T cells of the patient that haveleft the blood stream and have migrated into a tumor.

A further object of the present invention relates to a method forenhancing the potency of an immune checkpoint inhibitor administered toa subject as part of a treatment regimen, the method comprisingadministering to the subject a pharmaceutically effective amount of aSK1 inhibitor in combination with the immune checkpoint inhibitor.

As used herein the term “immune checkpoint protein” has its generalmeaning in the art and refers to a molecule that is expressed by T cellsin that either turn up a signal (stimulatory checkpoint molecules) orturn down a signal (inhibitory checkpoint molecules) Immune checkpointmolecules are recognized in the art to constitute immune checkpointpathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g.Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011.Nature 480:480-489). Examples of inhibitory checkpoint molecules includeA2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3and VISTA. The Adenosine A2A receptor (A2AR) is regarded as an importantcheckpoint in cancer therapy because the tumor microenvironment hasrelatively high levels of adenosine, which lead to a negative immunefeedback loop through the activation of A2AR. B7-H3, also called CD276,was originally understood to be a co-stimulatory molecule but is nowregarded as co-inhibitory. B7-H4, also called VTCN1, is expressed bytumor cells and tumor-associated macrophages and plays a role in tumorescape. B and T Lymphocyte Attenuator (BTLA), also called CD272, is aligand of HVEM (Herpesvirus Entry Mediator). Cell surface expression ofBTLA is gradually downregulated during differentiation of human CD8+ Tcells from the naive to effector cell phenotype, however tumor-specifichuman CD8+ T cells express high levels of BTLA. CTLA-4, CytotoxicT-Lymphocyte-Associated protein 4 and also called CD152 is overexpressedon Treg cells serves to control T cell proliferation. IDO, Indoleamine2,3-dioxygenase, is a tryptophan catabolic enzyme, a relatedimmune-inhibitory enzymes. Another important molecule is TDO, tryptophan2,3-dioxygenase. IDO is known to suppress T and NK cells, generate andactivate Tregs and myeloid-derived suppressor cells, and promote tumorangiogenesis. KIR, Killer-cell Immunoglobulin-like Receptor, is areceptor for MHC Class I molecules on Natural Killer cells. LAG3,Lymphocyte Activation Gene-3, works to suppress an immune response byaction to Tregs as well as direct effects on CD8+ T cells. PD-1,Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2.This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda,which gained FDA approval in September 2014. An advantage of targetingPD-1 is that it can restore immune function in the tumormicroenvironment. TIM-3, short for T-cell Immunoglobulin domain andMucin domain 3, expresses on activated human CD4+ T cells and regulatesTh1 and Th17 cytokines. TIM-3 acts as a negative regulator of Th1/Tc1function by triggering cell death upon interaction with its ligand,galectin-9. VISTA. Short for V-domain Ig suppressor of T cellactivation, VISTA is primarily expressed on hematopoietic cells so thatconsistent expression of VISTA on leukocytes within tumors may allowVISTA blockade to be effective across a broad range of solid tumors.

As used herein, the term “immune checkpoint inhibitor” has its generalmeaning in the art and refers to any compound inhibiting the function ofan immune inhibitory checkpoint protein. Inhibition includes reductionof function and full blockade. Preferred immune checkpoint inhibitorsare antibodies that specifically recognize immune checkpoint proteins. Anumber of immune checkpoint inhibitors are known and in analogy of theseknown immune checkpoint protein inhibitors, alternative immunecheckpoint inhibitors may be developed in the (near) future. The immunecheckpoint inhibitors include peptides, antibodies, nucleic acidmolecules and small molecules. In particular, the immune checkpointinhibitor of the present invention is administered for enhancing theproliferation, migration, persistence and/or cytoxic activity of CD8+ Tcells in the subject and in particular the tumor-infiltrating of CD8+ Tcells of the subject.

Thus the expression “enhancing the potency of an immune checkpoint”refers to the ability of the SK1 inhibitor to increase the ability ofthe immune checkpoint inhibitor to enhance the proliferation, migration,persistence and/or cytoxic activity of CD8+ T cells.

As used herein, the term “treatment” or “treat” refer to bothprophylactic or preventive treatment as well as curative or diseasemodifying treatment, including treatment of patient at risk ofcontracting the disease or suspected to have contracted the disease aswell as patients who are ill or have been diagnosed as suffering from adisease or medical condition, and includes suppression of clinicalrelapse. The treatment may be administered to a subject having a medicaldisorder or who ultimately may acquire the disorder, in order toprevent, cure, delay the onset of, reduce the severity of, or ameliorateone or more symptoms of a disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment. By “therapeutic regimen” is meant the pattern oftreatment of an illness, e.g., the pattern of dosing used duringtherapy. A therapeutic regimen may include an induction regimen and amaintenance regimen. The phrase “induction regimen” or “inductionperiod” refers to a therapeutic regimen (or the portion of a therapeuticregimen) that is used for the initial treatment of a disease. Thegeneral goal of an induction regimen is to provide a high level of drugto a patient during the initial period of a treatment regimen. Aninduction regimen may employ (in part or in whole) a “loading regimen”,which may include administering a greater dose of the drug than aphysician would employ during a maintenance regimen, administering adrug more frequently than a physician would administer the drug during amaintenance regimen, or both. The phrase “maintenance regimen” or“maintenance period” refers to a therapeutic regimen (or the portion ofa therapeutic regimen) that is used for the maintenance of a patientduring treatment of an illness, e.g., to keep the patient in remissionfor long periods of time (months or years). A maintenance regimen mayemploy continuous therapy (e.g., administering a drug at a regularintervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy(e.g., interrupted treatment, intermittent treatment, treatment atrelapse, or treatment upon achievement of a particular predeterminedcriteria [e.g., pain, disease manifestation, etc.]).

In some embodiments, the subject suffers from a cancer. As used herein,the term “cancer” has its general meaning in the art and includes, butis not limited to, solid tumors and blood-borne tumors. The term cancerincludes diseases of the skin, tissues, organs, bone, cartilage, bloodand vessels. The term “cancer” further encompasses both primary andmetastatic cancers. Examples of cancers that may be treated by methodsand compositions of the invention include, but are not limited to,cancer cells from the bladder, blood, bone, bone marrow, brain, breast,colon, esophagus, gastrointestinal tract, gum, head, kidney, liver,lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue,or uterus. In addition, the cancer may specifically be of the followinghistological type, though it is not limited to these: neoplasm,malignant; carcinoma; carcinoma, undifferentiated; giant and spindlecell carcinoma; small cell carcinoma; papillary carcinoma; squamous cellcarcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrixcarcinoma; transitional cell carcinoma; papillary transitional cellcarcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous; adenocarcinoma; mucoepidermoid carcinoma;cystadenocarcinoma; papillary cystadenocarcinoma; papillary serouscystadenocarcinoma; mucinous cystadenocarcinoma; mucinousadenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma;medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget'sdisease, mammary; acinar cell carcinoma; adenosquamous carcinoma;adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarianstromal tumor, malignant; thecoma, malignant; granulosa cell tumor,malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydigcell tumor, malignant; lipid cell tumor, malignant; paraganglioma,malignant; extra-mammary paraganglioma, malignant; pheochromocytoma;glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficialspreading melanoma; malignant melanoma in giant pigmented nevus;epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma;fibrous histiocytoma, malignant; myxosarcoma; liposarcoma;leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolarrhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerianmixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma;mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor,malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma;embryonal carcinoma; teratoma, malignant; struma ovarii, malignant;choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma,malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma;giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant;ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblasticfibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant;ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillaryastrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia.

In some embodiments, the subject suffers from melanoma. As used herein,“melanoma” refers to a condition characterized by the growth of a tumorarising from the melanocytic system of the skin and other organs. Mostmelanocytes occur in the skin, but are also found in the meninges,digestive tract, lymph nodes and eyes. When melanoma occurs in the skin,it is referred to as cutaneous melanoma. Melanoma can also occur in theeyes and is called ocular or intraocular melanoma. Melanoma occursrarely in the meninges, the digestive tract, lymph nodes or other areaswhere melanocytes are found. 40-60% of melanomas carry an activatingmutation in the gene encoding the serine-threonine protein kinase B-RAF(BRAF). Among the BRAF mutations observed in melanoma, over 90% are atcodon 600, and among these, over 90% are a single nucleotide mutationresulting in substitution of glutamic acid for valine (BRAFV600E).

In some embodiments, the subject suffers from a melanoma resistant toBRAF inhibitors. As used herein, the term “resistant” refers to therepeated outbreak of melanoma, or a progression of the melanomaindependently of whether the disease was cured before said outbreak orprogression. As used herein, the term “BRAF inhibitor” refers to anagent that is capable of inhibiting BRAF kinase or mutated BRAF kinaseactivity (one or more mutated forms of serine-threonine protein kinaseB-RAF (BRAF)) (e.g. BRAFV600E). Accordingly, the term “BRAF inhibitors”encompasses within its scope a compound that is capable of inhibitingBRAF or its mutated form; or a compound that is capable of inhibitingV600 mutated form of BRAF. Examples of BRAF inhibitors include but arenot limited to BAY43-9006 (sorafenib, Bayer), vemurafenib (PLX4032,Plexxikon; RG7204, RO5185426, Hofmann-LaRoche), GDC-0879(GlaxoSmithKline), dabrafenib (GSK21 18436, GlaxoSmithKline), PLX4720(Hofmann-LaRoche), BMS-908662 (XL281, Bristol-Myers Squibb), LGX818(Novartis), PLX3603 (RO5212054, Hofmann-LaRoche), ARQ-736 (ArQule),DP-4978 (Deciphera) or RAF265 (Novartis).

In some embodiments, the subject suffers from a melanoma with elevatedplasma lactate dehydrogenase (LDH). Plasma LDH can be considered“elevated” according to the method of the present invention if itexceeds plasma LDH levels typically found in a negative control, i.e., ahealthy mammal of the same species. Typically, plasma LDH can beconsidered “elevated” if it exceeds about 212 IU/mL. Preferably, plasmaLDH is considered “elevated” if it exceeds about 250 IU/mL. Morepreferably, plasma LDH is considered “elevated” if it exceeds about 287IU/mL.

Accordingly a further object of the present invention relates to amethod of treating cancer in a subject in need thereof comprisingadministering to the subject a therapeutically effective combination ofan immune checkpoint inhibitor with a SK1 inhibitor, whereinadministration of the combination results in enhanced therapeuticefficacy relative to the administration of the immune checkpointinhibitor alone.

As used herein, the expression “enhanced therapeutic efficacy,” relativeto cancer refers to a slowing or diminution of the growth of cancercells or a solid tumor, or a reduction in the total number of cancercells or total tumor burden. An “improved therapeutic outcome” or“enhanced therapeutic efficacy” therefore means there is an improvementin the condition of the patient according to any clinically acceptablecriteria, including, for example, decreased tumor size, an increase intime to tumor progression, increased progression-free survival,increased overall survival time, an increase in life expectancy, or animprovement in quality of life. In particular, “improved” or “enhanced”refers to an improvement or enhancement of 1%, 5%, 10%, 25% 50%, 75%,100%, or greater than 100% of any clinically acceptable indicator oftherapeutic outcome or efficacy. As used herein, the expression“relative to” when used in the context of comparing the activity and/orefficacy of a combination composition comprising the immune checkpointinhibitor with the SK1 inhibitor to the activity and/or efficacy of theimmune checkpoint alone, refers to a comparison using amounts known tobe comparable according to one of skill in the art.

In particular, the method of the present invention is particularlysuitable for the treatment of cancer characterized by a low tumorinfiltration of CD8+ T cells. Typically said tumor-infiltration of CD8+T cells is determined by any convention method in the art. For example,said determination comprises quantifying the density of CD8+ T cells ina tumor sample obtained from the subject.

As used herein, the term “tumor tissue sample” means any tissue tumorsample derived from the patient. Said tissue sample is obtained for thepurpose of the in vitro evaluation. In some embodiments, the tumorsample may result from the tumor resected from the patient. In someembodiments, the tumor sample may result from a biopsy performed in theprimary tumor of the patient or performed in metastatic sample distantfrom the primary tumor of the patient. For example an endoscopicalbiopsy performed in the bowel of the patient affected by a colorectalcancer. In some embodiments, the tumor tissue sample encompasses (i) aglobal primary tumor (as a whole), (ii) a tissue sample from the centerof the tumor, (iii) a tissue sample from the tissue directly surroundingthe tumor which tissue may be more specifically named the “invasivemargin” of the tumor, (iv) lymphoid islets in close proximity with thetumor, (v) the lymph nodes located at the closest proximity of thetumor, (vi) a tumor tissue sample collected prior surgery (for follow-upof patients after treatment for example), and (vii) a distantmetastasis. As used herein the “invasive margin” has its general meaningin the art and refers to the cellular environment surrounding the tumor.In some embodiments, the tumor tissue sample, irrespective of whether itis derived from the center of the tumor, from the invasive margin of thetumor, or from the closest lymph nodes, encompasses pieces or slices oftissue that have been removed from the tumor center of from the invasivemargin surrounding the tumor, including following a surgical tumorresection or following the collection of a tissue sample for biopsy, forfurther quantification of one or several biological markers, notablythrough histology or immunohistochemistry methods, through flowcytometry methods and through methods of gene or protein expressionanalysis, including genomic and proteomic analysis. The tumor tissuesample can, of course, be subjected to a variety of well-knownpost-collection preparative and storage techniques (e.g., fixation,storage, freezing, etc.). The sample can be fresh, frozen, fixed (e.g.,formalin fixed), or embedded (e.g., paraffin embedded).

In some embodiments, the quantification of density of CD8+ T cells isdetermined by immunohistochemistry (IHC). For example, thequantification of the density of CD8+ T cells is performed by contactingthe tissue tumor tissue sample with a binding partner (e.g. an antibody)specific for a cell surface marker of said cells. Typically, thequantification of density of CD8+ T cells is performed by contacting thetissue tumor tissue sample with a binding partner (e.g. an antibody)specific for CD8. Typically, the density of CD8+ T cells is expressed asthe number of these cells that are counted per one unit of surface areaof tissue sample, e.g. as the number of cells that are counted per cm²or mm² of surface area of tumor tissue sample. In some embodiments, thedensity of cells may also be expressed as the number of cells per onevolume unit of sample, e.g. as the number of cells per cm3 of tumortissue sample. In some embodiments, the density of cells may alsoconsist of the percentage of the specific cells per total cells (set at100%) Immunohistochemistry typically includes the following steps i)fixing the tumor tissue sample with formalin, ii) embedding said tumortissue sample in paraffin, iii) cutting said tumor tissue sample intosections for staining, iv) incubating said sections with the bindingpartner specific for the marker, v) rinsing said sections, vi)incubating said section with a secondary antibody typically biotinylatedand vii) revealing the antigen-antibody complex typically withavidin-biotin-peroxidase complex. Accordingly, the tumor tissue sampleis firstly incubated the binding partners. After washing, the labeledantibodies that are bound to marker of interest are revealed by theappropriate technique, depending of the kind of label is borne by thelabeled antibody, e.g. radioactive, fluorescent or enzyme label.Multiple labelling can be performed simultaneously. Alternatively, themethod of the present invention may use a secondary antibody coupled toan amplification system (to intensify staining signal) and enzymaticmolecules. Such coupled secondary antibodies are commercially available,e.g. from Dako, EnVision system. Counterstaining may be used, e.g. H&E,DAPI, Hoechst. Other staining methods may be accomplished using anysuitable method or system as would be apparent to one of skill in theart, including automated, semi-automated or manual systems. For example,one or more labels can be attached to the antibody, thereby permittingdetection of the target protein (i.e the marker). Exemplary labelsinclude radioactive isotopes, fluorophores, ligands, chemiluminescentagents, enzymes, and combinations thereof. In some embodiments, thelabel is a quantum dot. Non-limiting examples of labels that can beconjugated to primary and/or secondary affinity ligands includefluorescent dyes or metals (e.g. fluorescein, rhodamine, phycoerythrin,fluorescamine), chromophoric dyes (e.g. rhodopsin), chemiluminescentcompounds (e.g. luminal, imidazole) and bioluminescent proteins (e.g.luciferin, luciferase), haptens (e.g. biotin). A variety of other usefulfluorescers and chromophores are described in Stryer L (1968) Science162:526-533 and Brand L and Gohlke J R (1972) Annu. Rev. Biochem.41:843-868. Affinity ligands can also be labeled with enzymes (e.g.horseradish peroxidase, alkaline phosphatase, beta-lactamase),radioisotopes (e.g. ³H, ¹⁴C, ³²P, ³⁵S or ¹²⁵I) and particles (e.g.gold). The different types of labels can be conjugated to an affinityligand using various chemistries, e.g. the amine reaction or the thiolreaction. However, other reactive groups than amines and thiols can beused, e.g. aldehydes, carboxylic acids and glutamine Various enzymaticstaining methods are known in the art for detecting a protein ofinterest. For example, enzymatic interactions can be visualized usingdifferent enzymes such as peroxidase, alkaline phosphatase, or differentchromogens such as DAB, AEC or Fast Red. In other examples, the antibodycan be conjugated to peptides or proteins that can be detected via alabeled binding partner or antibody. In an indirect IHC assay, asecondary antibody or second binding partner is necessary to detect thebinding of the first binding partner, as it is not labeled. Theresulting stained specimens are each imaged using a system for viewingthe detectable signal and acquiring an image, such as a digital image ofthe staining. Methods for image acquisition are well known to one ofskill in the art. For example, once the sample has been stained, anyoptical or non-optical imaging device can be used to detect the stain orbiomarker label, such as, for example, upright or inverted opticalmicroscopes, scanning confocal microscopes, cameras, scanning ortunneling electron microscopes, canning probe microscopes and imaginginfrared detectors. In some examples, the image can be captureddigitally. The obtained images can then be used for quantitatively orsemi-quantitatively determining the amount of the marker in the sample.Various automated sample processing, scanning and analysis systemssuitable for use with immunohistochemistry are available in the art.Such systems can include automated staining and microscopic scanning,computerized image analysis, serial section comparison (to control forvariation in the orientation and size of a sample), digital reportgeneration, and archiving and tracking of samples (such as slides onwhich tissue sections are placed). Cellular imaging systems arecommercially available that combine conventional light microscopes withdigital image processing systems to perform quantitative analysis oncells and tissues, including immunostained samples. See, e.g., theCAS-200 system (Becton, Dickinson & Co.). In particular, detection canbe made manually or by image processing techniques involving computerprocessors and software. Using such software, for example, the imagescan be configured, calibrated, standardized and/or validated based onfactors including, for example, stain quality or stain intensity, usingprocedures known to one of skill in the art (see e.g., published U.S.Patent Publication No. US20100136549). The image can be quantitativelyor semi-quantitatively analyzed and scored based on staining intensityof the sample. Quantitative or semi-quantitative histochemistry refersto method of scanning and scoring samples that have undergonehistochemistry, to identify and quantitate the presence of the specifiedbiomarker (i.e. the marker). Quantitative or semi-quantitative methodscan employ imaging software to detect staining densities or amount ofstaining or methods of detecting staining by the human eye, where atrained operator ranks results numerically. For example, images can bequantitatively analyzed using a pixel count algorithms (e.g., AperioSpectrum Software, Automated QUantitatative Analysis platform (AQUA®platform), and other standard methods that measure or quantitate orsemi-quantitate the degree of staining; see e.g., U.S. Pat. Nos.8,023,714; 7,257,268; 7,219,016; 7,646,905; published U.S. PatentPublication No. US20100136549 and 20110111435; Camp et al. (2002) NatureMedicine, 8:1323-1327; Bacus et al. (1997) Analyt Quant Cytol Histol,19:316-328). A ratio of strong positive stain (such as brown stain) tothe sum of total stained area can be calculated and scored. The amountof the detected biomarker (i.e. the marker) is quantified and given as apercentage of positive pixels and/or a score. For example, the amountcan be quantified as a percentage of positive pixels. In some examples,the amount is quantified as the percentage of area stained, e.g., thepercentage of positive pixels. For example, a sample can have at leastor about at least or about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more positive pixels ascompared to the total staining area. In some embodiments, a score isgiven to the sample that is a numerical representation of the intensityor amount of the histochemical staining of the sample, and representsthe amount of target biomarker (e.g., the marker) present in the sample.Optical density or percentage area values can be given a scaled score,for example on an integer scale. Thus, in some embodiments, the methodof the present invention comprises the steps consisting in i) providingone or more immunostained slices of tissue section obtained by anautomated slide-staining system by using a binding partner capable ofselectively interacting with the marker (e.g. an antibody as abovedescribed), ii) proceeding to digitalisation of the slides of step a. byhigh resolution scan capture, iii) detecting the slice of tissue sectionon the digital picture iv) providing a size reference grid withuniformly distributed units having a same surface, said grid beingadapted to the size of the tissue section to be analyzed, and v)detecting, quantifying and measuring intensity of stained cells in eachunit whereby the number or the density of cells stained of each unit isassessed.

In some embodiments, the cell density of CD8+ T cells is determined inthe whole tumor tissue sample, is determined in the invasive margin orcentre of the tumor tissue sample or is determined both in the centreand the invasive margin of the tumor tissue sample.

Accordingly a further object of the present invention relates to amethod of treating cancer in a subject in need thereof comprising i)quantifying the density of CD8+ T cells in a tumor tissue sampleobtained from the subject ii) comparing the density quantified at stepi) with a predetermined reference value and iii) administering to thesubject a therapeutically effective combination of an immune checkpointinhibitor with a SK1 inhibitor when the density quantified at step i) islower than the predetermined reference value.

Typically, the predetermined reference value correlates with thesurvival time of the subject. Those of skill in the art will recognizethat OS survival time is generally based on and expressed as thepercentage of people who survive a certain type of cancer for a specificamount of time. Cancer statistics often use an overall five-yearsurvival rate. In general, OS rates do not specify whether cancersurvivors are still undergoing treatment at five years or if they'vebecome cancer-free (achieved remission). DSF gives more specificinformation and is the number of people with a particular cancer whoachieve remission. Also, progression-free survival (PFS) rates (thenumber of people who still have cancer, but their disease does notprogress) includes people who may have had some success with treatment,but the cancer has not disappeared completely. As used herein, theexpression “short survival time” indicates that the patient will have asurvival time that will be lower than the median (or mean) observed inthe general population of patients suffering from said cancer. When thepatient will have a short survival time, it is meant that the patientwill have a “poor prognosis”. Inversely, the expression “long survivaltime” indicates that the patient will have a survival time that will behigher than the median (or mean) observed in the general population ofpatients suffering from said cancer. When the patient will have a longsurvival time, it is meant that the patient will have a “goodprognosis”.

In some embodiments, the predetermined value is a threshold value or acut-off value. Typically, a “threshold value” or “cut-off value” can bedetermined experimentally, empirically, or theoretically. A thresholdvalue can also be arbitrarily selected based upon the existingexperimental and/or clinical conditions, as would be recognized by aperson of ordinary skilled in the art. For example, retrospectivemeasurement of cell densities in properly banked historical patientsamples may be used in establishing the predetermined reference value.The threshold value has to be determined in order to obtain the optimalsensitivity and specificity according to the function of the test andthe benefit/risk balance (clinical consequences of false positive andfalse negative). Typically, the optimal sensitivity and specificity (andso the threshold value) can be determined using a Receiver OperatingCharacteristic (ROC) curve based on experimental data. For example,after quantifying the density of CD8+ T cells in a group of reference,one can use algorithmic analysis for the statistic treatment of themeasured densities in samples to be tested, and thus obtain aclassification standard having significance for sample classification.The full name of ROC curve is receiver operator characteristic curve,which is also known as receiver operation characteristic curve. It ismainly used for clinical biochemical diagnostic tests. ROC curve is acomprehensive indicator that reflects the continuous variables of truepositive rate (sensitivity) and false positive rate (1-specificity). Itreveals the relationship between sensitivity and specificity with theimage composition method. A series of different cut-off values(thresholds or critical values, boundary values between normal andabnormal results of diagnostic test) are set as continuous variables tocalculate a series of sensitivity and specificity values. Thensensitivity is used as the vertical coordinate and specificity is usedas the horizontal coordinate to draw a curve. The higher the area underthe curve (AUC), the higher the accuracy of diagnosis. On the ROC curve,the point closest to the far upper left of the coordinate diagram is acritical point having both high sensitivity and high specificity values.The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, thediagnostic result gets better and better as AUC approaches 1. When AUCis between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracyis quite high. This algorithmic method is preferably done with acomputer. Existing software or systems in the art may be used for thedrawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statisticalsoftware, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS,CREATE-ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring,Md., USA), etc.

In some embodiments, the predetermined reference value is determined bycarrying out a method comprising the steps of a) providing a collectionof tumor tissue samples from subject suffering from the cancer ofinterest; b) providing, for each tumor tissue sample provided at stepa), information relating to the actual clinical outcome for thecorresponding patient (i.e. the duration of the disease-free survival(DFS) and/or the overall survival (OS)); c) providing a serial ofarbitrary quantification values; d) quantifying the density of CD8+ Tcells for each tumor tissue sample contained in the collection providedat step a); e) classifying said tumor tissue samples in two groups forone specific arbitrary quantification value provided at step c),respectively: (i) a first group comprising tumor tissue samples thatexhibit a quantification value for level that is lower than the saidarbitrary quantification value contained in the said serial ofquantification values; (ii) a second group comprising tumor tissuesamples that exhibit a quantification value for said level that ishigher than the said arbitrary quantification value contained in thesaid serial of quantification values; whereby two groups of tumor tissuesamples are obtained for the said specific quantification value, whereinthe tumor tissue samples of each group are separately enumerated; f)calculating the statistical significance between (i) the quantificationvalue obtained at step e) and (ii) the actual clinical outcome of thepatients from which tumor tissue samples contained in the first andsecond groups defined at step f) derive; g) reiterating steps f) and g)until every arbitrary quantification value provided at step d) istested; h) setting the said predetermined reference value as consistingof the arbitrary quantification value for which the highest statisticalsignificance (most significant) has been calculated at step g). Forexample the density of CD8+ T cells has been assessed for 100 tumortissue samples of 100 patients. The 100 samples are ranked according tothe density of CD8+ T cells. Sample 1 has the highest density and sample100 has the lowest density. A first grouping provides two subsets: onone side sample Nr 1 and on the other side the 99 other samples. Thenext grouping provides on one side samples 1 and 2 and on the other sidethe 98 remaining samples etc., until the last grouping: on one sidesamples 1 to 99 and on the other side sample Nr 100. According to theinformation relating to the actual clinical outcome for thecorresponding cancer patient, Kaplan Meier curves are prepared for eachof the 99 groups of two subsets. Also for each of the 99 groups, the pvalue between both subsets was calculated. The predetermined referencevalue is then selected such as the discrimination based on the criterionof the minimum p value is the strongest. In other terms, the density ofCD8+ T cells corresponding to the boundary between both subsets forwhich the p value is minimum is considered as the predeterminedreference value. It should be noted that the predetermined referencevalue is not necessarily the median value of cell densities. Thus insome embodiments, the predetermined reference value thus allowsdiscrimination between a poor and a good prognosis with respect to DFSand OS for a patient. Practically, high statistical significance values(e.g. low P values) are generally obtained for a range of successivearbitrary quantification values, and not only for a single arbitraryquantification value. Thus, in one alternative embodiment of theinvention, instead of using a definite predetermined reference value, arange of values is provided. Therefore, a minimal statisticalsignificance value (minimal threshold of significance, e.g. maximalthreshold P value) is arbitrarily set and a range of a plurality ofarbitrary quantification values for which the statistical significancevalue calculated at step g) is higher (more significant, e.g. lower Pvalue) are retained, so that a range of quantification values isprovided. This range of quantification values includes a “cut-off” valueas described above. For example, according to this specific embodimentof a “cut-off” value, the outcome can be determined by comparing thedensity of CD8+ T cells with the range of values which are identified.In some embodiments, a cut-off value thus consists of a range ofquantification values, e.g. centered on the quantification value forwhich the highest statistical significance value is found (e.g.generally the minimum p value which is found).

A further object of the present invention relates to a method oftreating cancer in a patient in need thereof comprising administering tothe patient a therapeutically effective amount of a SK-1 inhibitor incombination with a cancer vaccine.

As used herein, the term “cancer vaccine” has its general meaning in theart and refers to a composition capable of inducing active immunityagainst at least one cancer antigen. The cancer vaccine can result in aproduction of antibodies or simply in the activation of certain cells,in particular antigen-presenting cells, T lymphocytes (in particularT-CD8+ cells) and B lymphocytes. The cancer vaccine can be a compositionfor prophylactic purposes or for therapeutic purposes or both. As usedherein the term “antigen” refers to a molecule capable of beingspecifically bound by an antibody or by a T cell receptor (TCR) ifprocessed and presented by MHC molecules. The term “antigen”, as usedherein, also encompasses T-cell epitopes. An antigen is additionallycapable of being recognized by the immune system and/or being capable ofinducing a humoral immune response and/or cellular immune responseleading to the activation of B- and/or T-lymphocytes. An antigen canhave one or more epitopes or antigenic sites (B- and T-epitopes). Asused herein, the term “cancer antigen” refers to an antigen that ischaracteristic of a tumor tissue. There are multiple types of cancervaccines. Non-limiting examples of cancer vaccines include tumor cellvaccines, antigen vaccines, dendritic cell vaccines, DNA vaccines, andvector based vaccines.

Typically, the cancer vaccine of the present invention comprises atumor-associated antigen (“TAA”) or nucleic acid sequence (e.g. DNA)that encodes for a tumor-associated antigen. Numerous tumor-associatedantigens are known in the art. Exemplary tumor-associated antigensinclude, but are not limited to, 5 alpha reductase, alpha-fetoprotein,AM-1, APC, April, BAGE, beta-catenin, Bell 2, bcr-abl, CA-125,CASP-8/FLICE, Cathepsins, CD 19, CD20, CD21, CD23, CD22, CD33 CD35,CD44, CD45, CD46, CD5, CD52, CD55, CD59, CDC27, CDK4, CEA, c-myc, Cox-2,DCC, DcR3, E6/E7, CGFR, EMBP, Dna78, farnesyl transferase, FGF8b, FGF8a,FLK-1/KDR, folic acid receptor, G250, GAGE-family, gastrin 17,gastrin-releasing hormone, GD2/GD3/GM2, GnRH, GnTV, GP1, gp100/Pmel17,gp-100-in4, gp15, gp75/TRP-1, hCG, heparanse, Her2/neu, HMTV, Hsp70,hTERT, IGFR1, IL-13R, iNOS, Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA,LKLR-FUT, MAGE-family, mammaglobin, MAP 17, melan-A/MART-1, mesothelin,MIC A B, MT-MMPs, mucin, NY-ESO-1, osteonectin, p15, P170/MDR1, p53,p97/melanotransferrin, PAI-1, PDGF, uPA, PRAME, probasin,progenipoientin, PSA, PSM, RAGE-1, Rb, RCAS1, SART-1, SSX-family, STAT3,STn, TAG-72, TGF-alpha, TGF-beta, Thymosin-beta-15, TNF-alpha, TYRP-,TYRP-2, tyrosinase, VEGF, ZAG, pl6INK4, and glutathione-S-transferase.

In some embodiments, the vaccine is a DNA vaccine. Vectors can beengineered to contain specific DNAs that can be injected into a subjectwhich leads to the DNA being taken up by cells. Once the cells take upthe DNA, the DNA will program the cells to make specific antigens, whichcan then provoke the desired immune response.

In some embodiments, the vaccine consists of a recombinant virus thatencodes or express a cancer antigen. In some embodiments, therecombinant virus is a poxvirus expressing a tumor antigen and moreparticularly an orthopoxvirus such as, but not limited to, a vacciniavirus, a Modified Vaccinia Ankara (MVA) virus, or MVA-BN. Examples ofvaccinia virus strains are the strains Temple of Heaven, Copenhagen,Paris, Budapest, Dairen, Gam, MRIVP, Per, Tashkent, TBK, Tom, Bern,Patwadangar, BIEM, B-15, Lister, EM-63, New York City Board of Health,Elstree, Ikeda and WR. A preferred vaccinia virus (W) strain is theWyeth (DRYVAX) strain (U.S. Pat. No. 7,410,644). Another preferred Wstrain is a modified vaccinia virus Ankara (MVA) (Sutter, G. et al.[1994], Vaccine 12: 1032-40). Another preferred W strain is MVA-BN.Examples of MVA virus strains that are useful in the practice of thepresent invention and that have been deposited in compliance with therequirements of the Budapest Treaty are strains MVA 572, deposited atthe European Collection of Animal Cell Cultures (ECACC), VaccineResearch and Production Laboratory, Public Health Laboratory Service,Centre for Applied Microbiology and Research, Porton Down, Salisbury,Wiltshire SP4 OJG, United Kingdom, with the deposition number ECACC94012707 on Jan. 27, 1994, and MVA 575, deposited under ECACC 00120707on Dec. 7, 2000. MVA-BN, deposited on Aug. 30, 2000 at the EuropeanCollection of Cell Cultures (ECACC) under number V00083008, and itsderivatives, are additional exemplary strains. In some embodiments, theinvention encompasses the use of recombinant orthopoxviruses, preferablya vaccinia virus (W), a Wyeth strain, ACAM 1000, AC AM 2000, MVA, orMVA-BN for cancer therapy. Recombinant orthopoxviruses are generated byinsertion of heterologous sequences into an orthopoxvirus. In someembodiments, the recombinant poxvirus expressing a tumor antigen is anavipoxvirus, such as but not limited to a fowlpox virus. The term“avipoxvirus” refers to any avipoxvirus, such as Fowlpoxvirus,Canarypoxvirus, Uncopoxvirus, Mynahpoxvirus, Pigeonpoxvirus,Psittacinepoxvirus, Quailpoxvirus, Peacockpoxvirus, Penguinpoxvirus,Sparrowpoxviras, Starlingpoxviras and Turkeypoxviras. Preferredavipoxviruses are Canarypoxvirus and Fowlpoxvirus.

In some embodiments, the vaccine composition comprises at least onepopulation of antigen presenting cells that present the selectedantigen. The antigen-presenting cell (or stimulator cell) typically hasan MHC class I or II molecule on its surface, and in one embodiment issubstantially incapable of itself loading the MHC class I or II moleculewith the selected antigen. Preferably, the antigen presenting cells aredendritic cells. Suitably, the dendritic cells are autologous dendriticcells that are pulsed with the antigen of interest (e.g. a peptide).T-cell therapy using autologous dendritic cells pulsed with peptidesfrom a tumor associated antigen is disclosed in Murphy et al. (1996) TheProstate 29, 371-380 and Tjua et al. (1997) The Prostate 32, 272-278.Thus, in some embodiments, the vaccine composition containing at leastone antigen presenting cell is pulsed or loaded with one or moreantigenic peptides. As an alternative the antigen presenting cellcomprises an expression construct encoding an antigenic peptide. Thepolynucleotide may be any suitable polynucleotide and it is preferredthat it is capable of transducing the dendritic cell, thus resulting inthe presentation of a peptide and induction of an immune response.

In some embodiments, the vaccine composition include one or moreadjuvants. Adjuvants are substances that non-specifically enhance orpotentiate the immune response (e.g., immune responses mediated byCD8-positive T cells and helper-T (TH) cells to an antigen, and wouldthus be considered useful in the medicament of the present invention.Suitable adjuvants include, but are not limited to, 1018 ISS, aluminumsalts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellinor TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31,Imiquimod (ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2,IL-13, IL-21, Interferon-alpha or -beta, or pegylated derivativesthereof, IS Patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2,MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206,Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-wateremulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vectorsystem, poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

A further object of the present invention relates to a cancer vaccinecomprising an immunoadjuvant together with one or more cancer antigens,for inducing an immune response against said one or more cancer antigenswherein the immunoadjuvant is a SK-1 inhibitor.

The term “sphingosine kinase-1” or “SK1” refers to an enzyme thatcatalyzes the transformation of sphingosine to sphingosine-1-phosphate(S1P), i.e., phosphorylates sphingosine into S1P. Properties andactivities of SK1, e.g., protein sequence of SK1, structural propertiesof SK1, biochemical properties of SK1, and regulation of SK1, aredescribed in Taha et al. (2006, Journal of Biochemistry and MolecularBiology, 39(2): 113-131). Thus, as used herein the term “SK1 inhibitor”refers to any compound that is capable to inhibit SK1 expression oractivity. As used herein the term ‘SK1 activity” refers to theproduction, release, expression, function, action, interaction orregulation of SK1, including, e.g., temporal, site or distributionaspects. The activity of SK1 includes modifications, e.g., covalent ornon-covalent modifications of SK1 polypeptide, covalent or non-covalentmodifications that SK1 induces on other substances, changes in thedistribution of SK1 polypeptide, and changes that SK1 induces on thedistribution of other substances. Any aspect of SK1 activity can beevaluated. Methods and techniques known to those skilled in the art canbe found in various references, e.g., Ausubel et al., ed., CurrentProtocols in Mol. Biology, New York: John Wiley & Sons, 1990; Sambrooket al., Mol. Cloning, Cold Spring Harbor Laboratory Press, New York,N.Y. (1989). Examples of SK1 activity that can be evaluated includebinding activity of SK1 polypeptide to a binding molecule; the effect ofSK1 polypeptide on the posttranslational modification or stability of atarget gene; the level of SK1 protein; the level of SK1 mRNA; or thelevel of SK1 modification, e.g., phosphorylation, acetylation,methylation, carboxylation or glycosylation. By binding molecule ismeant any molecule to which SK1 can bind, e.g., a nucleic acid, e.g., aDNA regulatory region, a protein, a metabolite, a peptide mimetic, anon-peptide mimetic, an antibody, or any other type of ligand. Bindingcan be shown, e.g., by electrophoretic mobility shift analysis (EMSA),by the yeast or mammalian two-hybrid or three-hybrid assays, bycompetition with dimethylspingosine photoaffinity label or biotin-SK1binding. Transactivation of a target gene by SK1 can be determined,e.g., in a transient transfection assay in which the promoter of thetarget gene is linked to a reporter gene, e.g., β-galactosidase orluciferase, and co-transfected with a SK1 expression vector. Levels ofSK1 protein, mRNA or modification, can, e.g., be measured in a sample,e.g., a tissue sample, e.g., endothelial cells in blood vessels, T and Blymphocytes from blood or lymph organs, heart, muscle or bone joints. Insome embodiments, the evaluations are done in vitro; in otherembodiments the evaluations are done in vivo.

SK1 inhibitors are well known to the skilled person. For example theskilled person may easily identify such inhibitors from the followingpatent publications: WO2003105840, WO2006138660, WO2010033701,WO2010078247, WO2010127093, WO2011020116, WO2011072791, WO2012069852,WO2013119946, WO2014118556 and WO2014157382.

In some embodiments, the SK1 inhibitor is selected from the groupconsisting of 3-(4-Chloro-phenyl)-adamantane-1-carboxylic acidisopropylamide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylic acidcyclopropylamide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(2-ethylsulfanyl-ethyl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid phenylamide;Adamantane-1-carboxylic acid (4-hydroxy-phenyl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(4-hydroxy-phenyl)-amide; Acetic acid4-{[3-(4-chloro-phenyl)-adamantane-1-carbonyl]-amino}-phenyl ester;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(2,4-dihydroxy-phenyl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(3-hydroxymethyl-phenyl)-amide; Adamantane-1-carboxylic acid(4-cyanomethyl-phenyl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(4-cyanomethyl-phenyl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid4-tert-butyl-benzylamide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid 4-methylsulfanyl-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid3-trifluoromethyl-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid4-trifluoromethyl-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid3,5-bis-trifluoromethyl-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid3-fluoro-5-trifluoromethyl-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid2-fluoro-4-trifluoromethyl-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid3,5-difluoro-benzylamide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid 3,4-difluoro-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid3,4,5-trifluoro-benzylamide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid 3-chloro-4-fluoro-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid4-fluoro-3-trifluoromethyl-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid2-chloro-4-fluoro-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid4-chloro-3-trifluoromethyl-ben Σ ylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid3-aminomethyl-2,4,5,6-tetrachloro-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid[1-(4-chloro-phenyl)-ethyl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid[1-(4-bromo-phenyl)-ethyl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid4-methanesulfonyl-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid4-dimethylamino-benzylamide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid 4-trifluoromethoxy-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid3-trifluoromethoxy-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid 4-phenoxy-benzylamide;Adamantane-1-carboxylic acid 3,4-dihydroxy-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid3,4-dihydroxy-benzylamide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid phenethyl-amide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid[2-(4-fluoro-phenyl)-ethyl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid[2-(4-bromo-phenyl)-ethyl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid[2-(4-hydroxy-phenyl)-ethyl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid 4-phenoxy-benzylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid[2-(3-bromo-4-methoxy-phenyl)-ethyl]-amide; Adamantane-1-carboxylic acid[2-(3,4-dihydroxy-phenyl)-ethyl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid[2-(3,4-dihydroxy-phenyl)-ethyl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(2-benzo[1,3]dioxol-5-yl-ethyl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid[2-(3-phenoxy-phenyl)-ethyl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid[2-(4-phenoxy-phenyl)-ethyl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(3-phenyl-propyl)-amide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid (biphenyl-4-ylmethyl)-amide; Adamantane-1-carboxylic acid(1-methyl-piperidin-4-yl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(1-methyl-piperidin-4-yl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(4-methyl-piperazin-1-yl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(3-tert-butylamino-propyl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(3-pyrrolidin-1-yl-propyl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid[3-(2-oxo-pyrrolidin-1-yl)-propyl]-amide; Adamantane-1-carboxylic acid[2-(1-methyl-pyrrolidin-2-yl)-ethyl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid[2-(1-methyl-pyrrolidin-2-yl)-ethyl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(2-morpholin-4-yl-ethyl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(2-piperazin-1-yl-ethyl)-amide; Adamantane-1-carboxylic acid(pyridin-4-ylmethyl)-amide; 3-(4-Fluoro-phenyl)-adamantane-1-carboxylicacid (pyridin-4-ylmethyl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(pyridin-4-ylmethyl)-amide; Adamantane-1-carboxylic acid(pyridin-4-ylmethyl)-amide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid (2-pyridin-4-yl-ethyl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(3-imidazol-1-yl-propyl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(2-methyl-1H-indol-5-yl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(1H-tetrazol-5-yl)-amide; 3-(4-Chloro-phenyl)-adamantane-1-carboxylicacid (9-ethyl-9H-carbazol-3-yl)-amide; Adamantane-1-carboxylic acid[4-(4-chloro-phenyl)-thiazol-2-yl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid[4-(4-chloro-phenyl)-thiazol-2-yl]-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid benzothiazol-2-ylamide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid(5-chloro-benzooxazol-2-yl)-amide;3-(4-Chloro-phenyl)-adamantane-1-carboxylic acid (9H-purin-6-yl)-amide;[3-(4-Chloro-phenyl)-adamantane-1-ylmethyl]-isopropyl-amine4-{[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-amino}-phenol;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(4-trifluoromethyl-benzyl)-amine;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(2-fluoro-4-trifluoromethyl-benzyl)-amine;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(4-fluoro-3-trifluoromethyl-benzyl)-amine;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(4-trifluoromethoxy-benzyl)-amine;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-[2-(3-phenoxy-phenyl)-ethyl]-amine;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(1-methyl-piperidin-4-yl)-amine;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(4-methyl-piperazin-1-yl)-amine;N-tert-Butyl-N′-[3-(4-chloro-phenyl)-adamantan-1-ylmethyl]-propane-1,3-diamine;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(3-pyrrolidin-1-yl-propyl)-amine;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-[2-(1-methyl-pyrrolidin-2-yl)-ethyl]-amine;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(2-morpholin-4-yl-ethyl)-amine;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-pyridin-4-ylmethyl-amine;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-(9-ethyl-9H-carbazol-3-yl)-amine;[3-(4-Chloro-phenyl)-adamantan-1-ylmethyl]-[5-(4-chloro-phenyl)-thiazol-2-yl]-amine;1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethylamine;{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-isopropyl-amine;Phenyl-[1-(3-phenyl-adamantan-1-yl)-ethyl]-amine;{1-[3-(4-Fluoro-phenyl)-adamantan-1-yl]-ethyl}-ρhenyl-amine;{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-phenyl-amine;(1-Adamantan-1-yl-ethyl)-benzyl-amine;Benzyl-[1-(3-phenyl-adamantan-1-yl)-ethyl]-amine;Benzyl-{1-[3-(4-fluoro-phenyl)-adamantan-1-yl]-ethyl}-amine;Benzyl-{1-[3-(4-chloro-phenyl)-adamantan-1-yl]-ethyl}-amine;(4-tert-Butyl-benzyl)-{1-[3-(4-chloro-phenyl)-adamantan-1-yl]-ethyl}-amine;[1-(4-Bromo-phenyl)-ethyl]-{1-[3-(4-chloro-phenyl)-adamantan-1-yl]-ethyl}-amine;(1-Adamantan-1-yl-ethyl)-[2-(4-bromo-phenyl)-ethyl]-amine;[2-(4-Bromo-phenyl)-ethyl]-{1-[3-(4-chloro-phenyl)-adamantan-1-yl]-ethyl}-amine;(1-Adamantan-1-yl-ethyl)-(1-methyl-piperidin-4-yl)-amine;(1-Methyl-piperidin-4-yl)-[1-(3-phenyl-adamantan-1-yl)-ethyl]-amine;{1-[3-(4-Fluoro-phenyl)-adamantan-1-yl]-ethyl}-(1-methyl-piperidin-4-yl)-amine;{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(1-methyl-piperidin-4-yl)-amine;{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(4-methyl-piperazin-1-yl)-amine;{1-[3-(Phenyl)-adamantan-1-yl]-ethyl}-pyridin-4-ylmethyl-amine;{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(6-chloro-pyridin-3-ylmethyl)-amine;{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(2-pyridin-4-yl-ethyl)-amine;{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(3H-imidazol-4-ylmethyl)-amine;{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(2-methyl-1H-indol-5-yl)-amine;{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(9-ethyl-9H-carbazol-3-yl)-amine;{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(9-ethyl-9H-carbazol-3-ylmethyl)-amine;9-Ethyl-9H-carbazole-3-carboxylic acid{1-[3-(4-chloro-phenyl)-adamantan-1-yl]-ethyl}-amide;1-{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-3-(4-chloro-3-trifluoromethyl-phenyl)-urea;1-{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-3-(4-chloro-3-trifluoromethyl-phenyl)-urea;(4-Bromo-thiophen-2-ylmethyl)-{1-[3-(4-chloro-phenyl)-adamantan-1-yl]-ethyl}-amine;and{1-[3-(4-Chloro-phenyl)-adamantan-1-yl]-ethyl}-(4-phenyl-thiophen-2-ylmethyl)-amine

In some embodiments, the SK1 inhibitor of the present invention isselected from the group consisting of:

In some embodiments, the SK1 inhibitor isN′-[(2-hydroxynaphthalen-1-yl)methylidene]-3-(naphthalen-2-yl)-1H-pyrazole-5-carbohydrazidehaving the formula of:

In some embodiments, the SK1 inhibitor is an inhibitor of SK1expression. An “inhibitor of expression” refers to a natural orsynthetic compound that has a biological effect to inhibit theexpression of a gene. In a preferred embodiment of the invention, saidinhibitor of gene expression is a siRNA, an antisense oligonucleotide ora ribozyme. For example, anti-sense oligonucleotides, includinganti-sense RNA molecules and anti-sense DNA molecules, would act todirectly block the translation of SK1 mRNA by binding thereto and thuspreventing protein translation or increasing mRNA degradation, thusdecreasing the level of SK1, and thus activity, in a cell. For example,antisense oligonucleotides of at least about 15 bases and complementaryto unique regions of the mRNA transcript sequence encoding SK1 can besynthesized, e.g., by conventional phosphodiester techniques. Methodsfor using antisense techniques for specifically inhibiting geneexpression of genes whose sequence is known are well known in the art(e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323;6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) canalso function as inhibitors of expression for use in the presentinvention. SK1 gene expression can be reduced by contacting a subject orcell with a small double stranded RNA (dsRNA), or a vector or constructcausing the production of a small double stranded RNA, such that SK1gene expression is specifically inhibited (i.e. RNA interference orRNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of theinvention may be delivered in vivo alone or in association with avector. In its broadest sense, a “vector” is any vehicle capable offacilitating the transfer of the antisense oligonucleotide, siRNA, shRNAor ribozyme nucleic acid to the cells and typically cells expressingSK1. Typically, the vector transports the nucleic acid to cells withreduced degradation relative to the extent of degradation that wouldresult in the absence of the vector. In general, the vectors useful inthe invention include, but are not limited to, plasmids, phagemids,viruses, other vehicles derived from viral or bacterial sources thathave been manipulated by the insertion or incorporation of the antisenseoligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viralvectors are a preferred type of vector and include, but are not limitedto nucleic acid sequences from the following viruses: retrovirus, suchas moloney murine leukemia virus, harvey murine sarcoma virus, murinemammary tumor virus, and rous sarcoma virus; adenovirus,adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barrviruses; papilloma viruses; herpes virus; vaccinia virus; polio virus;and RNA virus such as a retrovirus. One can readily employ other vectorsnot named but known to the art.

In some embodiments, the immune checkpoint inhibitor is an antibodyselected from the group consisting of anti-CTLA4 antibodies, anti-PD-1antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies anti-TIM-3antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies.

As used herein, the term “antibody” is thus used to refer to anyantibody-like molecule that has an antigen binding region, and this termincludes antibody fragments that comprise an antigen binding domain suchas Fab′, Fab, F(ab′)2, single domain antibodies (DABs), TandAbs dimer,Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies,minibodies, diabodies, bispecific antibody fragments, bibody, tribody(scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody;kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager,scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domainantibody, bispecific format); SIP (small immunoprotein, a kind ofminibody); SMIP (“small modular immunopharmaceutical” scFv-Fc dimer;DART (ds-stabilized diabody “Dual Affinity ReTargeting”); small antibodymimetics comprising one or more CDRs and the like. The techniques forpreparing and using various antibody-based constructs and fragments arewell known in the art (see Kabat et al., 1991, specifically incorporatedherein by reference). Diabodies, in particular, are further described inEP 404,097 and WO 93/11161; whereas linear antibodies are furtherdescribed in Zapata et al. (1995). Antibodies can be fragmented usingconventional techniques. For example, F(ab′)2 fragments can be generatedby treating the antibody with pepsin. The resulting F(ab′)2 fragment canbe treated to reduce disulfide bridges to produce Fab′ fragments. Papaindigestion can lead to the formation of Fab fragments. Fab, Fab′ andF(ab′)2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies,diabodies, bispecific antibody fragments and other fragments can also besynthesized by recombinant techniques or can be chemically synthesized.Techniques for producing antibody fragments are well known and describedin the art. For example, each of Beckman et al., 2006; Holliger &Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al.,1996; and Young et al., 1995 further describe and enable the productionof effective antibody fragments. In some embodiments, the antibody ofthe present invention is a single chain antibody. As used herein theterm “single domain antibody” has its general meaning in the art andrefers to the single heavy chain variable domain of antibodies of thetype that can be found in Camelid mammals which are naturally devoid oflight chains. Such single domain antibody are also “nanobody®”. For ageneral description of (single) domain antibodies, reference is alsomade to the prior art cited above, as well as to EP 0 368 684, Ward etal. (Nature 1989 Oct. 12; 341 (6242): 544-6), Holt et al., TrendsBiotechnol., 2003, 21(11):484-490; and WO 06/030220, WO 06/003388.

In some embodiments, the antibody is a humanized antibody. As usedherein, “humanized” describes antibodies wherein some, most or all ofthe amino acids outside the CDR regions are replaced with correspondingamino acids derived from human immunoglobulin molecules. Methods ofhumanization include, but are not limited to, those described in U.S.Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and5,859,205, which are hereby incorporated by reference.

In some embodiments, the antibody is a fully human antibody. Fully humanmonoclonal antibodies also can be prepared by immunizing mice transgenicfor large portions of human immunoglobulin heavy and light chain loci.See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807,6,150,584, and references cited therein, the contents of which areincorporated herein by reference. These animals have been geneticallymodified such that there is a functional deletion in the production ofendogenous (e.g., murine) antibodies. The animals are further modifiedto contain all or a portion of the human germ-line immunoglobulin genelocus such that immunization of these animals will result in theproduction of fully human antibodies to the antigen of interest.Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAbmice (Medarex/GenPharm)), monoclonal antibodies can be preparedaccording to standard hybridoma technology. These monoclonal antibodieswill have human immunoglobulin amino acid sequences and therefore willnot provoke human anti-mouse antibody (KAMA) responses when administeredto humans. In vitro methods also exist for producing human antibodies.These include phage display technology (U.S. Pat. Nos. 5,565,332 and5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos.5,229,275 and 5,567,610). The contents of these patents are incorporatedherein by reference.

In some embodiments, the antibody comprises human heavy chain constantregions sequences but will not deplete CD8+ T cells to which they arebound and preferably do not comprise an Fc portion that induces antibodydependent cellular cytotoxicity (ADCC). As used herein, the term“depleting”, with respect to CD8+ T cells means a process, method, orcompound that can kill, eliminate, lyse or induce such killing,elimination or lysis, so as to negatively affect the number of CD8+ Tcells present in a sample or in a subject. The terms “Fc domain,” “Fcportion,” and “Fc region” refer to a C-terminal fragment of an antibodyheavy chain, e.g., from about amino acid (aa) 230 to about aa 450 ofhuman gamma heavy chain or its counterpart sequence in other types ofantibody heavy chains (e.g., α, δ, ε and μ for human antibodies), or anaturally occurring allotype thereof. Unless otherwise specified, thecommonly accepted Kabat amino acid numbering for immunoglobulins is usedthroughout this disclosure (see Kabat et al. (1991) Sequences of Proteinof Immunological Interest, 5th ed., United States Public Health Service,National Institute of Health, Bethesda, Md.). In some embodiments theantibody of the present invention does not lead, directly or indirectly,to the depletion of CD8+ T cells (e.g. do not lead to a 10%, 20%, 50%,60% or greater elimination or decrease in number CD8+ T cells). In someembodiments, the antibody of the present invention does not comprise anFc domain capable of substantially binding to a FcgRIIIA (CD16)polypeptide. In some embodiments, the antibody of the present inventionlacks an Fc domain (e.g. lacks a CH2 and/or CH3 domain) or comprises anFc domain of IgG2 or IgG4 isotype. In some embodiments, the antibody ofthe present invention consists of or comprises a Fab, Fab′, Fab′-SH, F(ab′) 2, Fv, a diabody, single-chain antibody fragment, or amultispecific antibody comprising multiple different antibody fragments.In some embodiments, the antibody of the present invention is not linkedto a toxic moiety. In some embodiments, one or more amino acids selectedfrom amino acid residues can be replaced with a different amino acidresidue such that the antibody has altered C2q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

Examples of anti-CTLA-4 antibodies are described in U.S. Pat. Nos:5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736;6,984,720; and 7,605,238. One anti-CTLA-4 antibody is tremelimumab,(ticilimumab, CP-675,206). In some embodiments, the anti-CTLA-4 antibodyis ipilimumab (also known as 10D1, MDX-D010) a fully human monoclonalIgG antibody that binds to CTLA-4.

Examples of PD-1 and PD-L1 antibodies are described in U.S. Pat. Nos.7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT PublishedPatent Application Nos: WO03042402, WO2008156712, WO2010089411,WO2010036959, WO2011066342, WO2011159877, WO2011082400, andWO2011161699. In some embodiments, the PD-1 blockers include anti-PD-L1antibodies. In certain other embodiments the PD-1 blockers includeanti-PD-1 antibodies and similar binding proteins such as nivolumab (MDX1106, BMS 936558, ONO 4538), a fully human IgG4 antibody that binds toand blocks the activation of PD-1 by its ligands PD-L1 and PD-L2;lambrolizumab (MK-3475 or SCH 900475), a humanized monoclonal IgG4antibody against PD-1; CT-011 a humanized antibody that binds PD-1;AMP-224 is a fusion protein of B7-DC; an antibody Fc portion; BMS-936559(MDX-1105-01) for PD-L1 (B7-H1) blockade.

Other immune-checkpoint inhibitors include lymphocyte activation gene-3(LAG-3) inhibitors, such as IMP321, a soluble Ig fusion protein(Brignone et al., 2007, J. Immunol. 179:4202-4211). Otherimmune-checkpoint inhibitors include B7 inhibitors, such as B7-H3 andB7-H4 inhibitors. In particular, the anti-B7-H3 antibody MGA271 (Loo etal., 2012, Clin. Cancer Res. July 15 (18) 3834). Also included are TIM3(T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcadeet al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J.Exp. Med. 207:2187-94). As used herein, the term “TIM-3” has its generalmeaning in the art and refers to T cell immunoglobulin and mucindomain-containing molecule 3. The natural ligand of TIM-3 is galectin 9(Gal9). Accordingly, the term “TIM-3 inhibitor” as used herein refers toa compound, substance or composition that can inhibit the function ofTIM-3. For example, the inhibitor can inhibit the expression or activityof TIM-3, modulate or block the TIM-3 signaling pathway and/or block thebinding of TIM-3 to galectin-9. Antibodies having specificity for TIM-3are well known in the art and typically those described in WO2011155607,WO2013006490 and WO2010117057.

In some embodiments, the immune checkpoint inhibitor is an IDOinhibitor. Examples of IDO inhibitors are described in WO 2014150677.Examples of IDO inhibitors include without limitation1-methyl-tryptophan (IMT), β-(3-benzofuranyl)-alanine,β-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6-fluoro-tryptophan,4-methyl-tryptophan, 5-methyl tryptophan, 6-methyl-tryptophan,5-methoxy-tryptophan, 5-hydroxy-tryptophan, indole 3-carbinol,3,3′-diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl1,3-diacetate, 9-vinylcarbazole, acemetacin, 5-bromo-tryptophan,5-bromoindoxyl diacetate, 3-Amino-naphtoic acid, pyrrolidinedithiocarbamate, 4-phenylimidazole a brassinin derivative, athiohydantoin derivative, a β-carboline derivative or a brassilexinderivative. Preferably the IDO inhibitor is selected from1-methyl-tryptophan, β-(3-benzofuranyl)-alanine, 6-nitro-L-tryptophan,3-Amino-naphtoic acid and β-[3-benzo(b)thienyl]-alanine or a derivativeor prodrug thereof.

As used herein the term “co-administering” as used herein means aprocess whereby the combination of the SK1 inhibitor and the immunecheckpoint inhibitor, is administered to the same patient. The SK1inhibitor and the immune checkpoint inhibitor may be administeredsimultaneously, at essentially the same time, or sequentially. Ifadministration takes place sequentially, the SK1 inhibitor isadministered before the immune checkpoint inhibitor. The SK1 inhibitorand the immune checkpoint inhibitor need not be administered by means ofthe same vehicle. The SK1 inhibitor and the immune checkpoint inhibitormay be administered one or more times and the number of administrationsof each component of the combination may be the same or different. Inaddition, the SK1 inhibitor and the immune checkpoint inhibitor need notbe administered at the same site.

As used herein, the term “therapeutically effective combination” as usedherein refers to an amount or dose of a SK1 inhibitor together with theamount or dose of the immune checkpoint inhibitor that is sufficient totreat the disease (e.g. cancer).The amount of the SK1 inhibitor in agiven therapeutically effective combination may be different fordifferent individuals and different tumor types, and will be dependentupon the one or more additional agents or treatments included in thecombination. The “therapeutically effective amount” is determined usingprocedures routinely employed by those of skill in the art such that an“improved therapeutic outcome” results. It will be understood, however,that the total daily usage of the compounds and compositions of thepresent invention will be decided by the attending physician within thescope of sound medical judgment. The specific therapeutically effectivedose level for any particular subject will depend upon a variety offactors including the disorder being treated and the severity of thedisorder; activity of the specific compound employed; the specificcomposition employed, the age, body weight, general health, sex and dietof the subject; the time of administration, route of administration, andrate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificpolypeptide employed; and like factors well known in the medical arts.For example, it is well within the skill of the art to start doses ofthe compound at levels lower than those required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved. However, the daily dosage of the productsmay be varied over a wide range from 0.01 to 1,000 mg per adult per day.Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0,10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient forthe symptomatic adjustment of the dosage to the subject to be treated. Amedicament typically contains from about 0.01 mg to about 500 mg of theactive ingredient, preferably from 1 mg to about 100 mg of the activeingredient. An effective amount of the drug is ordinarily supplied at adosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day,especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

According to the invention, the SK1 inhibitor and the immune checkpointinhibitor are administered to the subject in the form of apharmaceutical composition. Typically, the SK1 inhibitor and the immunecheckpoint inhibitor may be combined with pharmaceutically acceptableexcipients, and optionally sustained-release matrices, such asbiodegradable polymers, to form therapeutic compositions.“Pharmaceutically” or “pharmaceutically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type. Inthe pharmaceutical compositions of the present invention for oral,sublingual, subcutaneous, intramuscular, intravenous, transdermal, localor rectal administration, the active principle, alone or in combinationwith another active principle, can be administered in a unitadministration form, as a mixture with conventional pharmaceuticalsupports, to animals and human beings. Suitable unit administrationforms comprise oral-route forms such as tablets, gel capsules, powders,granules and oral suspensions or solutions, sublingual and buccaladministration forms, aerosols, implants, subcutaneous, transdermal,topical, intraperitoneal, intramuscular, intravenous, subdermal,transdermal, intrathecal and intranasal administration forms and rectaladministration forms. Typically, the pharmaceutical compositions containvehicles which are pharmaceutically acceptable for a formulation capableof being injected. These may be in particular isotonic, sterile, salinesolutions (monosodium or disodium phosphate, sodium, potassium, calciumor magnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions. The pharmaceutical forms suitablefor injectable use include sterile aqueous solutions or dispersions;formulations including sesame oil, peanut oil or aqueous propyleneglycol; and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In all cases, the form mustbe sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Solutions comprisingcompounds of the invention as free base or pharmacologically acceptablesalts can be prepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms. The SK1 inhibitorand the immune checkpoint inhibitor can be formulated into a compositionin a neutral or salt form. Pharmaceutically acceptable salts include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. The carrier can also be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), suitable mixtures thereof, and vegetables oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminium monostearate and gelatin. Sterileinjectable solutions are prepared by incorporating the active compoundsin the required amount in the appropriate solvent with several of theother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the typical methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. The preparation ofmore, or highly concentrated solutions for direct injection is alsocontemplated, where the use of DMSO as solvent is envisioned to resultin extremely rapid penetration, delivering high concentrations of theactive agents to a small tumor area. Upon formulation, solutions will beadministered in a manner compatible with the dosage formulation and insuch amount as is therapeutically effective. The formulations are easilyadministered in a variety of dosage forms, such as the type ofinjectable solutions described above, but drug release capsules and thelike can also be employed. For parenteral administration in an aqueoussolution, for example, the solution should be suitably buffered ifnecessary and the liquid diluent first rendered isotonic with sufficientsaline or glucose. These particular aqueous solutions are especiallysuitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration. In this connection, sterile aqueousmedia which can be employed will be known to those of skill in the artin light of the present disclosure. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1A-D. Downregulation of SK1 reduces melanoma tumor development inmice. (A), SK1 enzymatic activity was measured in Yumm cells stablytransfected with a control (shCtrl) or two SK1-targeted shRNA: shSK1 andshSK1(2). Enzyme activity (calculated as pmol/min/mg of proteins) incells transfected with shSK1 is compared to that of shCtrl cells. Dataare means±sem of 3 independent experiments. (B-D), shCtrl or shSK1 Yummcells (3.105) were injected in the dermis of wild-type (WT) C57BL/6 mice(B and C) or CD8-deficient mice (D). Tumor volume was determined at theindicated days (B and D) or at the end of the experiment (C, day 26).Growth profiles are presented as mean of tumor volume±SEM and arerepresentative of at least two independent experiments. (B and C n=12per group, D n=8 per group). Samples were compared using Mann-Whitneytest. *p<0.05, **p<0.01, ***p<0.001.

FIG. 2A-C. Downregulation of SK1 increases CD8+ T cell proliferation andactivation and inversely, reduces Treg. shCtrl or shSK1 Yumm murinemelanoma cells were injected in C57BL/6 mice and TILs were analyzed onday 11 by using flow cytometry. (A) CD8+, Foxp3+ CD4+ (Treg) and Foxp3−CD4+ T cells percentages and CD8+/Treg ratio in tumors at day 11. (B andC) The proportion of CD8+ (B) and Treg cells (C) expressing Ki67, PD-1and CTLA-4 was evaluated. Each symbol represents an independent tumor(n=9 mice per group). Results are representative of at least 2independent experiments. Samples were compared using Mann-Whitney test.*p<0.05, **p<0.01, ***p<0.001.

FIG. 3A-D. Downregulation of SK1 in tumor cells enhances the efficacy ofanti-CTLA-4 or anti-PD-1 therapy. Mice were challenged with 3.105 shCtrlor shSK1 Yumm cells on day 0, and then treated with control antibody(upper panels), α-CTLA-4 (days 7, 10 and 13) or/and α-PD-1 (days 5, 7and 10). (A and B) Individual curves are depicted for each tumor (n=6-11mice per group). Inserts: numbers indicate percentage of tumor-free miceat day 25. (C) Cumulative survival curves (Logrank test: *p<0.05;**p<0.01; ***p<0.001). (D) CD8/Treg ratio in tumors at day 11 arerepresented as Tukey box (n=10 per group). Results are representative ofat least 2 independent experiments. Samples were compared usingMann-Whitney test. *p<0.05, **p<0.01, ***p<0.001.

FIG. 4A-B. Pharmacological inhibition of SK1 synergizes with CTLA-4blockade to eradicate melanoma tumors. Mice were challenged with 3.105untransfected Yumm cells on day 0, and then treated or not with SKI-I ondays 5, 7, 10, 13 and 15. Control antibody or α-CTLA-4 was injected ondays 7, 10 and 13. (A) Tumor volumes determined at the indicated daysfor individual tumors are depicted (n=6 mice per group). Inserts:numbers indicate percentage of tumor-free mice at day 30. (B) Cumulativesurvival curves (Logrank test: *p<0.05).

FIG. 5A-B. Pharmacological inhibition of SK1 synergizes with anti-PD-1blockade to reduce tumor growth in melanoma and colon carcinoma. (A)Mice were challenged with 3.105 untransfected Yumm cells on day 0, andthen treated or not with SKI-I on days 5, 7, 10, 13 and 15. Controlantibody or α-PD-1 was injected on days 5, 7 and 10 (n=11-12 mice pergroup). (B) Mice were inoculated with 3.105 MC38 cells on day 0, andthen treated or not with SKI-I on days 5, 7, 10, 13 and 15. Controlantibody or α-PD-1 was injected on days 7 and 10 (n=4-6 mice per group).Tumor volume (means±sem for each group) was determined at the indicateddays. Samples were compared using Mann-Whitney test. *p<0.05.

EXAMPLE

Material & Methods

Cell Culture

Yumm murine melanoma cells, which harbor BRAFV600E mutation, Pten andCdkn2a deletion [1] were kindly provided by Dr. S. Tartare-Deckert(INSERM U1065 Nice, France). Yumm cells were grown as monolayers inOptiMEM media supplemented with 3% heat-inactivated fetal calf serum(FCS) in the presence of 5% CO2 in a humidified atmosphere at 37° C. Toguarantee cell line authenticity, Yumm cell lines were used for alimited number of passages and routinely tested for the expression ofmelanocyte-lineage proteins such as MelanA/MART1. MC38 cells were kindlyprovided by Drs T. Chardes et A. Pélegrin (INSERM U1194, Montpellier,France) and were cultured in DMEM containing 10% FCS, 2 mM glutamine,0.1 mM non essential amino acids, 1 mM sodium pyruvate and 10 mM Hepes.

Cell Transfection

Yumm cells were co-transfected, in a 1:10 ratio, with the pEGFP-N emptyvector and a SK1 shRNA (shSK1 or shSK1(2)) plasmid (shRNA fromThermoscientific) or a control non-targeting shRNA (shCtrl) plasmid(pLK01, Addgene). In brief, 500,000 cells were seeded in T25 cellculture flasks. Plasmids were diluted in OptiMEM (Thermofisher) mediumwithout serum. Cells were transfected with 10 μg shRNA oligomer usingLipofectamine 2000 reagent (Invitrogen) according to the manufacturer'sinstructions. Transfected cells were selected with 0.4 mg/ml G418 and1.5 μg/ml puromycin and GFP-expressing cells were sorted by FACS. Stabletransfectants were maintained in media containing 1 μg/ml puromycin; forthe experiments, cells were cultured in medium without puromycin.

SK1 Enzymatic Assay

SK1 activity was determined as described (Lavieu, Scarlatti et al. 2008)with minor modifications.

Tumor Cell Injections and Treatments in Mice

Animal experiments were conducted in accordance with national andinternational policies, and our protocol was approved by the RegionalEthics Committee of Midi-Pyrénées. 3.105 of Yumm cell lines(Untransfected, shCtrl, shSK1 or shSK1(2)) were intradermally injectedinto the flank of 7-week-old C57BL/6 mice (Charles River, L'Arbresle,France). CD8-deficient C57BL/6 mice were a gift from Prof. J. vanMeerwijk (INSERM U1043, Toulouse, France). Tumor volume was calculatedusing a caliper at the indicated days as described (Albinet, Bats et al.2014). For combination experiments involving shCtrl or shSK1 Yumm cells,mice were challenged intradermally (i.d.) with 3.105 cells on day 0 ontheir right flank. Mice were then injected i.p. three times withanti-CTLA-4 (200 μg per mouse on D7 and 100 μg per mouse on D10 andD13), and/or with anti-PD-1 or isotype control antibody (200 μg permouse on D5, D7 and D10). Tumor volumes were measured every 2-3 days.Anti-CTLA-4 (9H10), anti-PD-1 (RMP1-14) and isotype control (2A3) werepurchased from BioXcell.

For SKI-I treatment, 5 days after Yumm or MC38 cell implantation, micewere treated or not with 50 mg/kg SKI-I (N′-[(2-hydroxynaphthalen-1-yl)methylidene]-3-(naphthalen-2-yl)-1H-pyrazole-5-carbohydrazide, Enamine)in 50 μl of a mixture of DMSO (10%), Cremophor (5%), Tween-80 (5%) andglucose (80%) (i.p.). Mice received additional treatments of SKI-I ondays 7, 10, 13 and 15. Mice with Yumm tumors were injected i.p. withanti-CTLA-4 or anti-PD-1 or control antibody as described above. ForMC38 tumors, mice were injected i.p. two times with anti-PD-1 (100 μgper mouse on D7 and D10).

Analysis of Leukocyte Content in Tumors

Yumm cells (3.105) were intradermally injected into C57BL/6 mice. At day11, mice were sacrificed and tumors were collected and digested withMouse Tumor Dissociation kit and GentleMacs (Miltenyi). Cells werecounted and stained with the indicated antibodies and LIVE/DEAD reactivedyes (Invitrogen) prior to flow cytometry analysis (BD LSRFortessaX-20). Analyses were restricted to viable cells and performed usinganti-mouse CD45 (BD Biosciences), anti-mouse Thy1 (Biolegend),anti-mouse CD8 (Biolegend), anti-mouse CD4 (BD Biosciences), anti-mouseFoxp3 (eBioscience), anti-mouse Ki-67 (BD Bioscience), anti-mouse PD-1(eBioscience) or anti-mouse CTLA-4 (eBioscience). Isotype controls werefrom BD Biosciences, Biolegend or eBioscience.

Statistical Analyses

Data were analysed using GraphPad Prism (GraphPad Software, San Diego,Calif.). Results are expressed as means±sem. Student's t test was usedfor statistical comparisons among groups and differences were consideredstatistically significant when p<0.05 (*, p<0.05; **, p<0.01; ***,p<0.001). Tumor survival data were analyzed with the Kaplan-Meiermethod. The log-rank test was used to compare survival curves fordifferent subgroups on univariate analyses.

Results

SK1 Downregulation Reduces Tumor Growth and Enhances Antitumor Responsesto Melanoma

In order to evaluate the effect of SK1 in a syngeneic C57BL/6 mousemodel of melanoma, we used a transplantable tumor cell line (Yumm cells)established from a BrafV600E/+; Pten−/−; CDKN2A−/− mouse (Pencheva, Busset al. 2014). We generated stable SK1 knockdown Yumm cells, by using ashRNA-mediated silencing technology. We obtained two puromycin-resistantcell lines; shSK1 and shSK1(2), exhibiting a markedly reduced enzymaticactivity of SK1 (around 60% inhibition) (FIG. 1A). Then, Yumm cells, SK1knockdown or not (shCtrl) for SK1, were intradermally injected inC57BL/6 mice, and tumor growth was monitored. The tumor growth of shSK1and shSK1(2) Yumm cells was significantly lower than that of shCtrl Yummcells (FIGS. 1B and C). Interestingly, a tumor regression after day 11was observed in WT mice injected with ShSK1 cells that could reflect anincreased anti-melanoma immune response. However, this effect wasunlikely sufficient to obtain a long-lasting immune response, presumablydue to immune escape mechanisms. Importantly, SK1 knockdown failed toimpair Yumm melanoma growth in CD8-deficient mice (FIG. 1D).

To evaluate the impact of SK1 downregulation on the composition ofintratumoral lymphocyte infiltrate, we analyzed Tumor InfiltratingLymphocytes (TIL) on day 11. Of interest was the finding that SK1downregulation increased the proportion of CD8+ T cells and decreasedthe proportion of Foxp3+ CD4+ T cells (Treg) leading to a 4-foldincrease in CD8/Treg ratio (FIG. 2A). Moreover, the analysis of TILproliferation (as evaluated by monitoring Ki67 expression) andactivation (as evaluated by PD-1 and CTLA-4 expression) showed that SK1knockdown significantly increased CD8+ T cell proliferation andactivation (FIG. 2B) and inversely, decreased Treg proliferation as wellas CTLA-4 expression (FIG. 2C).

SK1 Downregulation Improves the Response to Immunotherapy

Given that SK1 downregulation was associated with an increase of tumoractivated CD8+ T cells, we hypothesized that SK1 inhibition may improvethe efficacy of Immune Checkpoint Inhibitors (ICI, e.g., anti-CTLA-4 andanti-PD-1). As shown in FIG. 3, whereas anti-CTLA-4 or/and anti-PD-1treatment alone had limited effects on established Yumm tumors (FIG.3A), SK1 silencing dramatically enhanced the response to anti-CTLA-4 oranti-PD-1 treatment, leading to tumor rejection in 100% and 67% of theanimals, respectively (FIG. 3B). Moreover, SK1 downregulationsignificantly improved overall survival (FIG. 3C). Indeed, thiscombination (ICI+SK1 silencing) induced durable cures in 100% and 42% ofthe mice treated with anti-CTLA-4 and anti-PD-1, respectively, 2 monthsafter cessation of therapy, suggesting the establishment of an effectiveimmunological memory. Interestingly, amongst the long-term survivors,none of them developed a tumor upon a second melanoma cell injection,indicating that they were fully vaccinated against this melanoma cellline (data not shown). This enhanced response to ICI was associated withan increased CD8/Treg ratio in tumors (FIG. 3D). Of note, the CD8/Tregratio is impressively increased in the tumors of Yumm ShSK1+anti-CTLA-4group (Fold Change=16), this could explain the total tumor regressionobserved when using this combination.

Synergistic Anti-Cancer Immune Response of Immune Checkpoint Blockadeand SK1 Pharmacological Inhibition.

To further confirm the potency of the combined therapy based on SK1downregulation and ICI, we used SKI-I, a pharmacological inhibitor ofSK1 (French, Schrecengost et al. 2003). Our data show that, whereasCTLA-4 blockade alone led to no tumor rejection at all, the combinationof SKI-I+anti-CTLA-4 greatly synergized, resulting to total rejection in67% of mice (FIG. 4A) and improved the overall survival (FIG. 4B). Toconfirm our observation with anti-PD-1, mice harboring Yumm tumors weretreated with SKI-I combined or not with anti-PD-1. As shown in FIG. 5A,SKI-I enhanced the efficacy of anti-PD-1. Importantly, this effect wasalso observed in mice inoculated with MC38 colon carcinoma (FIG. 5B).

Collectively our data indicate that greater therapeutic success will beachieved by combining immune checkpoint blockade with agents thatmodulate the oncogenic SK1/S1P pathway. Interfering with sphingolipidmetabolism may facilitate the development of novel avenues fortherapeutic intervention in melanoma as well as in other cancer types.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

Albinet, V., M. L. Bats, A. Huwiler, P. Rochaix, C. Chevreau, B. Segui,T. Levade and N. Andrieu-Abadie (2014). “Dual role of sphingosinekinase-1 in promoting the differentiation of dermal fibroblasts and thedissemination of melanoma cells.” Oncogene 33(26): 3364-3373.

French, K. J., R. S. Schrecengost, B. D. Lee, Y. Zhuang, S. N. Smith, J.L. Eberly, J. K. Yun and C. D. Smith (2003). “Discovery and evaluationof inhibitors of human sphingosine kinase.” Cancer Res 63(18):5962-5969.

Lavieu, G., F. Scarlatti, G. Sala, S. Carpentier, T. Levade, R. Ghidoni,J. Botti and P. Codogno (2008). “Sphingolipids in macroautophagy.”Methods Mol Biol 445: 159-173.

Pencheva, N., C. G. Buss, J. Posada, T. Merghoub and S. F. Tavazoie(2014). “Broad-spectrum therapeutic suppression of metastatic melanomathrough nuclear hormone receptor activation.” Cell 156(5): 986-1001.

1. A method for enhancing the potency of an immune checkpoint inhibitoradministered to a subject as part of a treatment regimen, the methodcomprising administering to the subject a pharmaceutically effectiveamount of a SK1 inhibitor in combination with the immune checkpointinhibitor.
 2. A method of treating cancer in a subject in need thereofcomprising administering to the subject a therapeutically effectivecombination of an immune checkpoint inhibitor with a SK1 inhibitor,wherein administration of the combination results in enhancedtherapeutic efficacy relative to the administration of the immunecheckpoint inhibitor alone.
 3. The method of claim 1 wherein the subjectsuffers from a cancer selected from the group consisting of neoplasm,malignant; carcinoma; carcinoma, undifferentiated; giant and spindlecell carcinoma; small cell carcinoma; papillary carcinoma; squamous cellcarcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrixcarcinoma; transitional cell carcinoma; papillary transitional cellcarcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous; adenocarcinoma; mucoepidermoid carcinoma;cystadenocarcinoma; papillary cystadenocarcinoma; papillary serouscystadenocarcinoma; mucinous cystadenocarcinoma; mucinousadenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma;medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget'sdisease, mammary; acinar cell carcinoma; adenosquamous carcinoma;adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarianstromal tumor, malignant; thecoma, malignant; granulosa cell tumor,malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydigcell tumor, malignant; lipid cell tumor, malignant; paraganglioma,malignant; extra-mammary paraganglioma, malignant; pheochromocytoma;glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficialspreading melanoma; malignant melanoma in giant pigmented nevus;epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma;fibrous histiocytoma, malignant; myxosarcoma; liposarcoma;leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolarrhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerianmixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma;mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor,malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma;embryonal carcinoma; teratoma, malignant; struma ovarii, malignant;choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma,malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma;giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant;ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblasticfibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant;ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillaryastrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia.
 4. The method of claim 1 wherein the subject suffers froma melanoma.
 5. The method of claim 1 wherein the subject suffers from amelanoma resistant to melanoma resistant to BRAF inhibitors.
 6. Themethod of claim 1 wherein the subject suffers from a melanoma withelevated plasma dehydrogenase (LDH).
 7. The method of claim 1 whereinthe cancer is characterized by a low tumor infiltration of CD8+ T cells.8. The method of claim 1 wherein the SK1 inhibitor is selected from thegroup consisting of:


9. The method of claim 1 wherein the SK1 inhibitor isN′-[(2-hydroxynaphthalen-1-yl)methylidene]-3-(naphthalen-2-yl)-1H-pyrazole-5-carbohydrazide.10. The method of claim 1 wherein the SK1 inhibitor is an inhibitor ofSK1 expression.
 11. The method of claim 1 wherein the immune checkpointinhibitor is an antibody selected from the group consisting ofanti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies,anti-PD-L2 antibodies anti-TIM-3 antibodies, anti-LAG3 antibodies,anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, andanti-B7H6 antibodies.
 12. A method of treating cancer in a subject inneed thereof comprising i) quantifying the density of CD8+ T cells in atumor tissue sample obtained from the subject ii) comparing the densityquantified at step i) with a predetermined reference value and iii)administering to the subject a therapeutically effective combination ofan immune checkpoint inhibitor with a SK1 inhibitor when the densityquantified at step i) is lower than the predetermined reference value.